The elimination of activated T cells is important to maintain homeostasis and avoid immunopathology. CD95 (Fas/APO-1) has been identified as a death mediator for activated T cells in vitro but the function of CD95 in death of mature T cells in vivo is still controversial. Here we show that triggering of the costimulatory TNF receptor family member CD27 sensitized T cells for CD95-induced apoptosis. CD95-deficient (lpr/lpr) T cells massively expanded and differentiated into IFN-γ-secreting effector cells in transgenic mice that constitutively express the CD27 ligand, CD70. Concomitantly, CD95-deficient CD70 transgenic mice became moribund by 4 wk of age with severe liver pathology and bone marrow failure. These findings establish that CD95 is a critical regulator of effector T cell homeostasis in chronic immune activation.
After initial clonal expansion in response to Ag most of the responding T cells die by apoptosis and only few Ag-specific T cells are preserved as memory T cells. The death of the activated T cells is important to maintain homeostasis and avoid immunopathology generated by toxic side effects of effector T cell molecules (1).
Signaling through members of the death domain containing TNF receptor (TNFR) family such as CD95, TNFR1, and DR4/5 induces apoptosis by the recruitment of a signal transduction complex that activates caspase enzymes (2). Active caspases induce cleavage of structural and regulatory proteins within cells and leads ultimately to apoptosis. Humans and mice with mutations in the genes encoding for either CD95 or its ligand progressively accumulate lymphocytes in the periphery and are susceptible to develop autoimmune diseases (2). In vitro CD95 was identified as an essential mediator of apoptosis for activated T cells (3, 4, 5). Subsequent studies using mice deficient in CD95 (lpr mice) or CD95 ligand (gld mice) showed that CD95-CD95L interactions regulate T cell homeostasis (6, 7, 8, 9). However, other reports suggested that the death of primed T cells in vivo is independent from the CD95 system (10, 11, 12, 13).
TNFR-associated factor (TRAF)2 binding TNFR family members, such as OX40, 4–1BB, and CD27, influence T cell division, survival, and effector function (14). These receptors can antagonize mitochondrion-dependent cell death by up-regulating anti-apoptotic Bcl-2 family molecules such as Bcl-xL and Bfl (Refs. 15 and 16 and unpublished data).
In the present study, we questioned whether CD95-induced cell death has a role in the regulation of expansion of T cells stimulated via CD27. Whereas previous reports showed that CD27 stimulation enhanced the magnitude of T cell responses (17, 18), we here found that CD27-CD70 interaction sensitizes T cells for CD95-mediated apoptosis, thereby providing evidence for an in-built feedback mechanism. Thus, CD27-CD70 interactions can operate as a double-edged sword by either inducing T cell survival while increasing sensitivity to death by CD95.
Materials and Methods
The mice strains used were C57BL/6, CD70 Tg (21), CD27−/− (17), F5 TCR Tg (30) (kindly provided by Dr. D. Kioussis, National Institute for Medical Research, London, U.K.), IFN-γ−/−, and lpr/lpr mice (purchased from The Jackson Laboratory). All mice strains are on a C57BL/6 background. CD70 Tg mice were crossed with CD27−/−, F5 TCR Tg, lpr/lpr, and IFN-γ−/− mice. All mice were bred in the animal facility of The Netherlands Cancer Institute (Amsterdam) under specific pathogen-free conditions and were handled in accordance with institutional and national guidelines. CD27−/−, IFN-γ−/−, and lpr/lpr mutant mice were genotyped by PCR analysis of tail DNA. CD70 Tg and F5 TCR Tg mice were genotyped by FACS analysis using CD70 mAb and Vβ11 mAb, respectively.
Cell preparation and flow cytometry
Single cell suspensions were prepared from freshly isolated spleens, lymph nodes (axillary, brachial, inguinal, and mesenteric), and thymus by mincing and gently pressing the tissues through cell strainers. Bone marrow (femurs and tibias) single cell suspensions were prepared by flushing the bones with a needle. Erythrocytes were lysed with ammonium chloride buffer. Surface staining and intracellular staining for cell surface molecules and cytokines was performed as described (21) using Abs to CD4, CD8, CD62L, CD44, CD45R/B220, CD43 (clone 1B11), CD95, IL-2, TNF, IFN-γ, and IL-10 (BD Pharmingen). Flow cytometric analysis was performed on a FACSCalibur with Cell Quest software (BD Biosciences).
To purify wild-type T cells, lymph node cell suspensions from 6- to 8-wk-old wild-type mice were incubated with rat-anti-mouse MHC class II and rat-anti-mouse B220 Abs for 30 min at 4°C, washed and incubated with goat-anti-rat IgG microbeads (Miltenyi Biotec) for 20 min at 4°C. After washing, cells were magnetically separated with the MACS (negative selection T cells). To purify B cells, splenic cell suspensions from CD70 Tg and CD27−/− mice were incubated with mouse CD19 microbeads (Miltenyi Biotec) for 20 min at 4°C. After washing, cells were magnetically separated with the MACS (positive selection B cells). The T and B cell fractions were consistently found to be >95% CD3+ and >95% B220+, respectively, as evidenced by flow cytometry.
T cell activation, CD95 expression, and CD95-mediated apoptosis assay
T cells (1 × 106 cells/ml) were stimulated with 1 μg/ml coated CD3 mAb (purified from hybridoma supernatants, clone CRL 1975) and cultured 1:1 with either purified CD27−/− × CD70 Tg B cells or purified CD27−/− B cells in IMDM medium containing 10% FCS. CD95 expression on T cells was analyzed after 24 h by flow cytometry with PE-conjugated anti-CD95 mAb (clone Jo2; BD Pharmingen) and allophycocyanin-conjugated antiCD3 mAb (BD Pharmingen). For CD95-mediated apoptosis-purified hamster anti-mouse CD95 mAb (5 μg/106 cells, clone Jo2; BD Pharmingen) was added, and after 48 h, cells were subjected to flow cytometric analysis. Propidium iodide was used to identify apoptotic cells and allophycocyanin-conjugated anti-CD3 mAb to identify T cells.
For the adoptive transfers, 8 × 106 enriched T cells from spleen and LN cells (>75% Vβ11+CD8+ T cell) of TCR Tg and lpr/lpr× TCR Tg were injected i.v. into either CD27+/− or CD27−/−× CD70 Tg recipients. Two days after cellular transfer, recipient mice were infected with 25 hemagglutination unit influenza virus. At day 6 and day 11 after viral challenge, the expansion of the donor population was quantitated in spleen by flow cytometry using Abs to CD8 and Vβ11. The donor population was distinguished from the endogenous Vβ11 expressing CD8+ T cells by difference in fluorescence intensity.
Histological analysis and immunohistochemistry
Tissues were fixed in 4% phosphate-buffered formalin, embedded in paraffin, sectioned at 4 μm, and stained with H&E according to standard procedures. For immunohistochemistry, tissues were deparaffinized in xylene and rehydrated. Endogenous peroxidase activity was blocked by using 3% (v/v) H2O2 in methanol for 10 min. Before staining, paraffin sections were pretreated by heat-induced epitope retrieval. Slides were incubated with 5% normal goat serum/PBS for 30 min, and subsequently sections were incubated overnight with a 1:400 dilution of CD3 or B220 at 4°C. mAb immunoreactivity was detected with the streptavidin-biotin immunoperoxidase (sABC) method by using biotinylated goat anti-rat IgG (Dako, 1:100) as secondary antibody, and diaminobenzidine substrate for visualization. After counterstaining with hematoxylin, slides were mounted. For negative control, the primary mAb was omitted.
Results and Discussion
CD27 stimulation sensitizes T cells for CD95-mediated apoptosis
We first tested whether CD27 stimulation by its ligand CD70 influenced CD95 cell surface expression on T cells and/or modulated susceptibility to CD95-induced apoptosis. CD3-stimulated wild-type T cells were cultured in the presence or absence of CD27 stimulation by the addition of CD70 transgenic (Tg) or control B cells, respectively. CD95 expression on T cells increased with CD27 stimulation (Fig. 1,A). Moreover, an increased proportion of the CD3/CD27 stimulated T cells underwent CD95-induced apoptosis compared with CD3 treatment alone (Fig. 1,B), indicating that CD70 sensitizes T cells for CD95-mediated apoptosis in vitro. To test CD95 susceptibility in vivo, we adoptively transferred influenza-specific TCR Tg T cells and CD95-deficient (lpr/lpr) influenza-specific TCR Tg T cells into either CD27−/−or CD27−/−× CD70 Tg mice. The number of influenza-specific T cells was assessed 6 and 11 days after infection of the recipient mice with influenza virus. CD95 deficiency did not affect the size of the influenza-specific population in the spleen when T cells were transferred into CD27−/− animals but strongly enhanced T cell expansion in CD70 Tg × CD27−/− mice (Fig. 1 C). Thus, in analogy to what has been shown for CD40 signaling in the B cell system (19, 20), CD27 signaling appeared to protect Ag-expanded T cells from passive cell death but concomitantly primed these cells for CD95-mediated deletion.
Neonatal death of CD95-deficient CD70 Tg mice
To assess the importance of the CD95 system in an in vivo model for chronic T cell activation, CD95-deficient (lpr/lpr) mice were bred with CD70 Tg mice. In CD70 Tg mice, constitutive CD27 triggering by CD70 on B cells causes a progressive conversion of naive T cells into effector-memory cells, which ultimately results in exhaustion of the immune system and lethal immunodeficiency as seen in persisting, active viral infections (21, 22). CD95-deficient CD70 Tg animals were born with an expected Mendelian frequency and looked healthy at birth. However, after 3 wk the CD95-deficient CD70 Tg mice became sick and died at around 4 wk of age (Fig. 2,A). In contrast, CD70 Tg mice with normal CD95 expression died at around 25 wk of age. The spleen and lymph nodes (LN) of CD95-deficient CD70 Tg mice had a normal size but histology revealed that normal splenic and LN architecture was disrupted (Fig. 2,B and data not shown). Furthermore, a severe atrophy of the thymus was observed, and diffuse T lymphocytic infiltrates were seen in the liver but not in heart, kidney, lungs, or gut. A strong decreased cellularity of the hematopoietic lineages was seen in bone marrow (Fig. 2 B).
CD95 constrains effector T cell formation in CD70 Tg mice
Concerning the composition of the peripheral lymphoid compartments, the spleens of 2-wk-old CD95-deficient CD70 Tg mice contained normal T cell numbers whereas in LN a weakly reduced T cell pool was observed (Fig. 3, A and B). Strikingly, the percentage of activated effector T cells (defined by a CD62lowCD44brightCD43high phenotype) was increased compared with the various control animals (wild type, CD70 Tg, and lpr/lpr). Comparison of 2-wk-old with 3-wk-old mice showed that the accumulation of effector T cells was progressive and was most prominent in the CD8+ T cell subset (Fig. 3, A and B). In agreement with the enhanced effector T cell formation, splenic and LN CD4+ and CD8+ T cells from CD95-deficient CD70 TG mice had a strong increase in the ability to synthesize IFN-γ and IL-10 and a somewhat reduced capacity to produce TNF and IL-2 (Fig. 3,C and data not shown). Finally, the progressive B cell depletion observed in CD70 Tg mice (21) was greatly accelerated in CD95-deficient CD70 Tg mice (Fig. 2,B and Fig. 3 D).
Expression of TCR-Vβ elements did not differ between CD95-deficient CD70 Tg mice and controls (data not shown), which suggests that, as in CD70 Tg mice (22), innocuous environmental or autoantigens may drive effector T cell formation. In support of this, crossing CD95-deficient CD70 Tg mice with Tg mice expressing an MHC class I-restricted influenza-specific F5 TCR, restrained CD8+ effector T cell formation whereas in these animals the CD4+ compartment largely consisted of effector-type T cells (Fig. 3,E). As an apparent consequence of the reduced effector T cell formation in comparison to CD95-deficient CD70 Tg, premature death was postponed to around 12 wk of age in CD95-deficient CD70 Tg × TCR Tg mice (Fig. 2 A). Thus, CD95 deficiency in mice with a chronically activated immune system results in excessive Ag-induced effector T cell formation and leads to premature death.
Effects of IFN-γ in CD70-driven effector T cell formation and immunopathology
Suppressor of cytokine signaling (SOCS-1)−/− mice have an exaggerated response to IFN-γ and develop a complex neonatal disease (23) that has, with respect to bone marrow and liver pathology, conspicuous similarities to the pathology observed in CD95-deficient CD70 Tg mice. Previously, we have shown that IFN-γ is responsible for progressive B cell depletion in CD70 Tg mice (21). Since the formation of IFN-γ-producing effector T cells is strongly enhanced in CD95-deficient CD70 Tg mice, we crossed CD95-deficient CD70 Tg mice on an IFN-γ background to test whether IFN-γ is responsible for the pathology in these mice. The survival of CD95-deficient CD70 Tg × IFN-γ−/− mice was slightly improved compared with CD95-deficient CD70 Tg mice (3.9 ± 0.5 wk CD95-deficient CD70 Tg vs 6 ± 1.1 wk CD95-deficient CD70 Tg × IFN-γ−/−) (Fig. 4,A). Remarkably, histological analysis revealed that the pathology was completely distinct. The size of spleens and LN of CD95-deficient CD70 Tg × IFN-γ−/− mice were substantially larger than of CD95-deficient CD70 Tg and CD70 Tg × IFN-γ−/− mice. Moreover, focal perivascular infiltrates of lymphocytic blasts were found in liver, lung and kidney (Fig. 4,B and data not shown). B cell numbers in CD95-deficient CD70 Tg × IFN-γ−/− mice were only slightly higher than in CD95-deficient CD70 Tg mice (2.2 × 106± 0.6 × 106 CD95-deficient CD70 Tg vs 3.1 × 106± 1.5 × 106 CD95-deficient CD70 Tg × IFN-γ−/− per spleen; 0.27 × 106± 0.21 × 106 CD95-deficient CD70 Tg vs 0.6 × 106± 0.12 × 106 CD95-deficient CD70 Tg × IFN-γ−/− for LN), indicating an IFN-γ independent B cell reduction in these mice. T cell numbers, notably CD8+ T cells, in spleen and LN were strongly increased with an overrepresentation of effector-type T cells (Fig. 4 C). These T cells produced, as in CD95-deficient CD70 Tg mice, levels of TNF and IL-2 that were somewhat reduced compared with control mice. However, IL-10 secretion was not detectable in these T cells (data not shown). The massive expansion of T cells would be in line with the suggested function of IFN-γ in promoting the contraction of effector T cell populations (24, 25), which may at least partly be explained by facilitating CD95-mediated apoptosis (26). The difference in pathology between IFN-γ-competent and IFN-γ-deficient mice infer that although excessive IFN-γ has detrimental effects on liver and bone marrow functions also other pathological changes related to disturbed homeostasis of effector T cells are associated with mortality.
The mechanism of priming for CD95-induced cell death by CD27 signaling is unknown. A direct effect on inhibition of passive cell death is unlikely since Bcl-2 Tg T cells are not protected from apoptosis by CD95 (27). IL-2 has been described as a key regulator of effector T cell homeostasis by blocking passive cell death and promoting death receptor-induced apoptosis (28). It is unlikely that IL-2 is a key player for the observations in CD95-deficient CD70 Tg mice since neither IL-2 production (Fig. 3 C) nor expression of the IL-2 receptor (data not shown) was significantly altered in these mice. Rather, it seems that multiple independent signaling pathways that generate extensive expansion of immune cells at the same time sensitize these cells for death receptor-mediated deletion thereby providing a negative feedback mechanism controlling unlimited growth and persistence.
In acute immune responses to infection, the Bcl-2 homologue protein Bim was found to be an important mediator for the death of activated T cells (13, 29). We here found that CD95-mediated apoptosis is crucial to maintain homeostasis of effector T cells in a situation where the immune system is chronically activated via costimulatory TNFR family members that signal via TRAF molecules, such as CD27. Repeated triggering of these TNFRs may occur in situations of persisting pathogens since many of the TNFR ligands are activation molecules that are up-regulated by Ag-specific activation (14). Thus homeostasis of effector T cells is dependent on the proper balance between signals provided by TRAF-binding and death domain-containing TNFR family members.
We thank the staff of the Animal Facility of The Netherlands cancer Institute for excellent animal care.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Abbreviations used in this paper: TRAF, TNFR-associated factor; Tg, transgenic; LN, lymph nodes.